Study Of CD137L Prokaryotic Expression,Purification And Function

Objective:The prokaryotic expression system of CD137L recombinant protein was constructed and the expression parameters of CD137L recombinant plasmid expression bacteria were optimized.The CD137L recombinant protein was purified and renatured.Then the biological function of the recombinant protein was studied by combined immunization with HPV16 Vaccine in TC-1 tumor model mice.Methods:The codon-optimized CD137L gene was amplified and sub-cloned to construct the recombinant T plasmid pGEM/CD137L,which was digested by restriction enzyme Ndel and Xhol double enzyme.The enzyme-digested product was inserted into the pET30a vector to build the recombinant expression plasmid pET30a/CD137L.After identification by PCR and enzyme digestion,the recombinant expression plasmid was transformed to the BL21(DE3)for expression.To optimize the expression of the recombinant CD137L protein,the following expression parameters were considered:(1)pH medium conditions;(2)induction time;(3)KANA concentration;(4)inoculation rate;(5)inducer concentration;(6)inducing temperature.The optimization expression parameters were used to product a large number of recombinant proteins for purification by affinity chromatography and renaturation.At last,the TC-1 tumor model mice were combined immunization by the purified protein and HPV16 Vaccine.Results:Through 6 experiments,preliminary engineering strain was finally obtained,the optimum culture and expression parameters were as follows:(1)pH medium conditions:7.09;(2)induction time:OD600 = 1.1;(3)KANA concentration:50mg/L;(4)inoculation amount:5%;(5)concentration of inducer 0.25mM;(6)inducing temperature:37 degrees.The inclusion protein of CD137L was purified by affinity chromatography,and refolded in buffer(20mM Glycine pH9.0,0.5%Tween-80,5%Glycerol,lmmol/L DTT).Biological function of CD137L recombinant protein was studied by TC-1 tumor model mice,the tumor model mice was subjected to CD137L and HPV16 recombinant protein vaccine.The results showed that the mice rate of negative control,CD137L 1?g,CD137L 5?g,CD137L 50?g group were less than 10%,and independent HPV vaccine could hold-up 40%model mice with no tumor growing.CD137L1?g and CD137L5p,g could raise the tumor inhibition level of HPV vaccine to 90%.In the HPV vaccine plus CD137L50?g group,the mice tumor model inhibition rate was 50%.Conclusion:Through the experimental study,the recombinant expression bacteria of CD137L protein was constructed and screened.Through the experiment of 6 conditions,the optimum culture and expression condition of the engineering strain was finally obtained.And recombinant protein of CD137L expressed in such condition can be used to improve the immune effect of human papillomavirus type 16(HPV16)recombinant protein vaccine after purification and dialysis.